Mitochondrial fission during mitophagy requires both inner and outer mitofissins

  1. Kentaro Furukawa
  2. Tatsuro Maruyama
  3. Shun-ichi Yamashita
  4. Keiichi Inoue
  5. Tomoyuki Fukuda
  6. Nobuo N. Noda
  7. Tomotake Kanki

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Abstract

Mitophagy maintains mitochondrial homeostasis through selective degradation of damaged or excess mitochondria. Recently, we identified mitofissin/Atg44, a mitochondrial intermembrane space-resident fission factor, which directly acts on lipid membranes and drives mitochondrial fission required for mitophagy in yeast. However, it remains unclear whether mitofissin is sufficient for mitophagy-associated mitochondrial fission and whether other factors act from outside the mitochondria. Here, we identify a mitochondrial outer membrane-resident mitofissin-like microprotein required for mitophagy, and we name it mitofissin 2/Mfi2 based on the following results. Overexpression of a C-terminally truncated form of Mfi2 induces mitochondrial fragmentation and partially restores mitophagy in atg44 Δ cells. Mfi2 binds to lipid membranes and mediates membrane fission in vitro , demonstrating its intrinsic mitofissin activity. Genetic analyses reveal that Mfi2 and the dynamin-related protein Dnm1 independently facilitate mitochondrial fission during mitophagy. Thus, Atg44 and Mfi2, two mitofissins with distinct localizations, are required for mitophagy-associated mitochondrial fission.

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  1. Review Commons

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    Reply to the reviewers

    Manuscript number: RC-2025-03004

    Corresponding author(s): Kentaro Furukawa and Tomotake Kanki

    1. General Statements [optional]

    We would like to thank the reviewers for their constructive and positive feedback. We are encouraged that all three reviewers consider the identification of Mfi2 as an outer mitochondrial membrane fission factor required for mitophagy to be a significant and important contribution to the research field. We acknowledge the concerns raised and propose the following plan to address them through additional experiments and clarifications. We believe that these revisions will further strengthen the manuscript and enhance its impact.

    2. Description of the planned revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Furukawa and colleagues identified Mfi2 as novel factor that promotes fragmentation and removal of damaged mitochondria by mitophagy. They report that parallel loss of Dnm1 and Mfi2 blocks mitophagy. Mfi2 acts on the outer membrane, while the previous found Atg44 functions in the intermembrane space. How the proteins cooperate remains unknown. This is an elegant study with high-quality data. The findings are interesting for a broad readership. There are some issues as outline below that should be solved.

    Response:

    We would like to thank Reviewer #1 for their thoughtful evaluation of our manuscript and for recognizing the interest and quality of the study.

    It remains unclear how Mfi2 is anchored into the outer mitochondrial membrane. Does it contain a transmembrane domain? The carbonate resistance indicates the presence of such transmembrane domain. However, the presented structures lack such membrane-spanning segment. This point should be clarified.

    Response:

    We performed an in silico topology prediction of Atg44 and Mfi2 using TMHMM. This tool identified a weakly hydrophobic region of Mfi2 near the N-terminus but did not predict a definitive transmembrane domain (see new Fig. EV1E) (Page 6, lines 8-9). This result implies that Mfi2 interacts with the outer membrane in a monotopic or peripheral manner, rather than as a classical transmembrane protein. Such proteins may remain in the membrane pellet after carbonate treatment due to their strong hydrophobic insertion into the lipid bilayer (e.g., yeast tafazzin/Taz1; Brandner et al., Mol. Biol. Cell, 2005; DOI: 10.1091/mbc.E05-03-0256). We will incorporate this interpretation in the revised manuscript.

    How does Mfi2 cooperate with Dnm1? Is there any interaction between these proteins? Some further information could provide mechanistic insights into the function of Mfi2.

    Response:

    While our study does not explicitly suggest that Mfi2 cooperates with Dnm1, we plan to investigate whether these proteins physically associate. We will perform co-immunoprecipitation experiments under growing and mitophagy-inducing conditions to examine potential interactions between Mfi2 and Dnm1. Further insights into their interaction could help clarify the mechanistic role of Mfi2 in mitochondrial fission and mitophagy.

    The authors report a CL-dependent binding of Mfi2 to liposomes. Is the recruitment of Mfi2 to mitochondria impaired when CL-synthesis is blocked, e.g. in crd1delta mitochondria?

    Response:

    To assess the role of cardiolipin in Mfi2 localization, we will compare the efficiency of mitochondrial targeting of endogenous Mfi2 in WT and crd1Δ cells. Additionally, as mentioned in Reviewer #3's comment, we plan to perform coarse-grained molecular dynamics simulations to further investigate the interaction between Mfi2 and cardiolipin. The results of these simulations will be incorporated into the discussion to provide deeper mechanistic insights.

    Figure 4B: a wild-type control should be added.

    Response:

    We appreciate Reviewer #1’s suggestion to include a WT control in Figure 4B. However, given the focus of this figure on the rescue of mitophagy defects in the mfi2Δ dnm1Δ strain, we believe that adding a WT control is not essential for the analysis. The key comparison here is between the mfi2Δ dnm1Δ strain and the rescue conditions, and statistical analysis was performed to support the conclusions. We hope this clarifies our approach, but we will make adjustments if necessary.

    Reviewer #1 (Significance (Required)):

    The reported findings are interesting for a broad readership.

    Response:

    We appreciate Reviewer #1’s recognition of the relevance of our findings to a broad readership.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this study, the authors discover a mitochondrial fission factor, termed Mfi2, that promotes mitophagy efficiency and that functions in a partially redundant way with Dnm1 for the fission of mitochondrial outer membranes during mitophagy. The discovery helps to clarify why Dnm1 does not appear to be essential for fission mediated mitophagy by Dnm1. Mfi2 is structurally similar to the inner membrane fission factor Atg44 which is consistent with Mfi2's fission activity. The authors show that Mfi2 has membrane fission activity towards nanotubes in vitro, and that membrane binding is dependent of high levels of cardiolipin, a mitochondrially enriched lipid. In summary, the authors show that Mfi2 mediates mitochondrial outer membrane fission together with Drp1, whereas Atg44 mediates inner membrane fission, that together are necessary for mitophagy.

    Response:

    We thank Reviewer #2 for the positive assessment and for clearly summarizing the main contributions of our work.

    Major:

    1. Figure 2: How do the expression levels of the Mfi2 constructs compare to the endogenous levels of the protein? This will help to gauge to what degree Mfi2 N66 overexpression is needed to achieve mitochondrial fragmentation in Atg44 delta cells and also the low level of mitophagy rescue that was observed.

    Response:

    We used the TDH3 promoter for the expression of Mfi2 in Figures 2D and 2E. Unfortunately, our Mfi2 antibody only detects full-length Mfi2, as it recognizes a C-terminal region of the protein. This means we cannot directly compare the expression levels of Mfi2(N66) to those of endogenous full-length Mfi2. To clarify the expression levels, we will provide the following data:

    (1) Mfi2 antibody: Endogenous Mfi2(Full) and overexpressed Mfi2(Full)

    (2) FLAG antibody: Overexpressed Mfi2(Full)-FLAG and overexpressed Mfi2(N66)-FLAG

    Figure 3A-B: The cardiolipin binding results in vitro are interesting but the concentration of cardiolipin is much lower on the outer membrane versus the inner membrane. Can the authors comment on whether the cardiolipin levels used on the nanotubes are relevant to that of the mitochondrial outer membrane where Mfi2 is located? Can the authors provide quantitative data for these experiments to help strengthen their conclusions?

    Can the authors also use purified MBP alone or a form of Mfi2 that cannot bind to membrane e.g. Mfi2-C33) as a control?

    Response:

    We thank the reviewer for raising this important point regarding our cardiolipin-dependent in vitro data. In our experiments, we used 20 mol% cardiolipin (CL), a concentration higher than the typical levels in the mitochondrial outer membrane, which contains less than 5% CL. However, it is known that CL translocates to the outer membrane under mitophagy-inducing conditions (e.g., Chu et al., Nat Cell Biol, 2013; Kagan et al., Cell Death Differ, 2016). Our use of elevated CL levels aligns with standard practices in in vitro reconstitution assays to ensure adequate membrane curvature and charge density, which are necessary for robust and reproducible protein-membrane interaction assessments.

    To strengthen our conclusions, we will provide a quantitative analysis of the nanotube fission experiments. This will include the percentage of severed tubes under each condition, the total number of tubes analyzed (n), and the relationship between tube diameter and fission efficiency. These additional data will allow for a more thorough evaluation of the membrane fission activity of Mfi2.

    Furthermore, we will include control experiments using purified MBP alone and a membrane-binding-deficient mutant of Mfi2 (C33), as suggested by the reviewer.

    Figure 4D: The protrusions are very difficult to visualize. Can the authors also provide zoomed in regions. Is the data representative from 3 or more independent experiments? Can the authors provide a graph of the quantitation to aid readers with analysis of the data?

    Response:

    We thank the reviewer for this helpful suggestion. In the revised manuscript, we will provide higher magnification images to improve the visibility of mitochondrial protrusions. We confirm that the presented images are representative of results obtained from three independent experiments. Additionally, as requested, we will include a graph quantifying the frequency and morphology of protrusions to facilitate data interpretation.

    Figure 4D: It is fascinating to see the mitochondrial protrusion formation being dependent on autophagy factors but not mitochondrial fission factors. To help visualize this, can the authors image one of either Atg1, Atg8 to address whether phagophores are forming on the protrusions and if so where they are positionally located on the protrusion in control and/or mfi2,dnm1,atg44 triple mutant cells?

    Response:

    We thank the reviewer for this insightful comment. In our previous study (Fukuda et al., Mol Cell, 2023), we demonstrated that Atg proteins, such as Atg8, accumulate at mitochondrial protrusions formed in atg44Δ cells, suggesting that these structures can serve as sites for phagophore assembly. However, as in our previous microscopy analysis, the resolution limitations of our imaging system make it difficult to precisely determine the exact location of phagophores on the protrusions.

    Whether similar recruitment occurs in the absence of both Mfi2 and Dnm1 remains untested. To address this, we will perform fluorescence imaging of fluorescent protein tagged Atg proteins, such as GFP-Atg8, in mfi2Δ dnm1Δ atg44Δ triple mutant cells to examine whether phagophores form on the mitochondrial protrusions under these conditions. This will help us determine whether phagophore formation requires mitochondrial fission or occurs independently of it.

    Minor:

    1. Is it possible to target Atg44 to the mitochondrial outer membrane, either by attaching an OM anchor or using part of the N-terminus of Mfi2? This will help elucidate how Mfi2 reaches the outer membrane and whether Atg44 can be just as active on the outer membrane as long as it can access it.

    Response:

    We thank the reviewer for this suggestion. We will construct chimeric proteins between Atg44 and Mfi2 and examine where such proteins are localized. Additionally, we will assess whether these chimeric proteins have the functional activity of Mfi2, as this will help determine if Atg44 can be active on the mitochondrial outer membrane when properly targeted.

    Are microtubules or actin required for the protrusions to form? Using the triple mutant cells that have a high proportion of protrusions, it could be tried to add cytoskeletal depolymerizing drugs such as nocodazole for microtubules or Latrunculin A or Latrunculin B for actin.

    Response:

    We thank the reviewer for this suggestion. We will test the effect of cytoskeletal depolymerizing drugs on protrusion formation in the mfi2Δ dnm1Δ atg44Δ triple mutant cells.

    Reviewer #2 (Significance (Required)):

    Significance: The discovery of Mfi2 as an outer membrane mitophagy fission factor is an exciting, and very important and significant contribution to the field. The data are in this study are clear and the conclusions are generally well supported by the experiments. This study appears to be suitable as a report style manuscript given that there is limited mechanistic analysis of Mfi2 activity. This does not affect the importance of the work, it just means that it is suited as a report of a significant discovery. Overall, this fills an important knowledge gap in solving the mystery behind which factors are involved in mitochondrial outer membrane fission during mitophagy, and provides a clarification why Dnm1 loss alone minimally affects mitophagy. This work will appeal to researchers interested in mitochondrial biology, the autophagy field, and cell biologists interested in organelle membrane dynamics, and is also broadly important and interesting to all cell biologists.

    Reviewer expertise: mitophagy mechanisms, cell biology of mitophagy, autophagy and autophagosome formation, mitochondrial biology including OXPHOS and mitochondrial dynamics

    Response:

    We appreciate Reviewer #2’s comments on the importance and potential impact of our discovery for the mitophagy and cell biology fields.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Furukawa et al. presents a well-structured and thorough study identifying Mfi2 as a novel mitochondrial outer membrane-resident fission factor required for mitophagy in Saccharomyces cerevisiae. The authors demonstrate that Mfi2, together with the inner membrane mitofissin Atg44 and the dynamin-related GTPase Dnm1, contributes to mitochondrial fragmentation during mitophagy. Importantly, they show that while Dnm1 is dispensable on its own, Mfi2 and Dnm1 act redundantly from the outer membrane to support Atg44-mediated fission. The data are robust, the figures are clear, and the mechanistic insight into how mitophagy-specific fission is achieved is of high relevance to the field of mitochondrial quality control.

    Overall, this is a logically constructed and convincing study with important implications for understanding compartment-specific mechanisms of mitochondrial fission during selective autophagy. The conclusions are largely well supported by the data. However, a few issues and points of clarification should be addressed before publication.

    Response:

    We thank Reviewer #3 for the careful and constructive review and for acknowledging the logical structure and robustness of our data.

    Major Comments

    1. The observation that both Mfi2 and Atg44 require high cardiolipin (CL) content for membrane binding and fission in vitro is intriguing, especially given that CL is enriched in the inner membrane. The authors mention CL externalisation during mitophagy, but this connection could be made more explicit earlier in the manuscript. Furthermore, since the molecular mechanism of membrane interaction remains unresolved, I would strongly encourage the authors to undertake coarse-grained molecular dynamics simulations to explore how Mfi2 might interact with lipid bilayers of differing composition. This could clarify the role of CL and the potential structural contribution of the disordered C-terminal region. Response:

    We thank the reviewer for highlighting the need to clarify the connection between CL externalization and the observed CL-dependent membrane binding and fission activity of Mfi2 and Atg44. While we briefly mentioned CL externalization during mitophagy in the Discussion, we agree that this connection should be made more explicit earlier in the manuscript. In the revised version, we will incorporate a brief rationale in the Results section to clarify that CL translocates to the mitochondrial outer membrane under mitophagy-inducing conditions (e.g., Chu et al., Nat Cell Biol 2013). This will provide a physiological basis for our in vitro reconstitution assays using CL-containing liposomes.

    We also appreciate the reviewer’s suggestion to explore the molecular basis of Mfi2-lipid interaction through coarse-grained molecular dynamics (CGMD) simulations. In collaboration with Dr. Yuji Sakai, we will perform coarse-grained molecular dynamics (CGMD) simulations to investigate how Mfi2 interacts with lipid bilayers of varying compositions, focusing particularly on the role of cardiolipin and the structural contribution of the disordered C-terminal region. If successful, we will include the results in the revised manuscript.

    While the genetic and phenotypic data indicate that Mfi2 and Dnm1 act independently to support mitochondrial fission, the spatial and temporal organisation of their activity during mitophagy remains unclear. Do Mfi2 and Dnm1 colocalise at fission sites, or do they act at separate subdomains of the outer membrane? Live-cell imaging with fluorescently tagged Mfi2 and Dnm1, particularly during mitophagy induction, could help clarify whether these factors act in concert or at distinct locations and time points. This would also help determine whether their apparent redundancy reflects parallel mechanisms or functional compensation at shared sites. It would also be interesting to combine this with Atg44.

    Response:

    We thank the reviewer for this insightful comment. We plan to perform co-localization analysis of Mfi2 and Dnm1 during mitophagy induction to clarify whether these proteins colocalize at fission sites or act at separate subdomains of the outer membrane. Additionally, we will conduct co-immunoprecipitation experiments of Mfi2 and Dnm1 (see also Response to Reviewer #1’s major comment 2) to further investigate their potential interaction. It is challenging to analyze Mfi2, Dnm1, Atg44, and mitochondrial fission sites simultaneously, as fluorescence-tagged Atg44 has been shown to lose its function (Fukuda et al., Mol Cell, 2023).

    Minor Comments

    1. The sodium carbonate extraction and proteinase K assays (Figure 1E-F) are standard but may not be familiar to all readers. A brief explanatory sentence clarifying what these methods reveal about membrane topology would improve accessibility. Response:

    We thank the reviewer for this helpful comment. We have added a brief explanatory sentence in the revised manuscript to clarify the principles and interpretation of the sodium carbonate extraction and proteinase K assays (Page 5, lines 23-25; Page 6, lines 1-3).

    While immunoblot quantifications are shown throughout, it would be helpful to include statistical analysis where appropriate, especially in cases where differences between genotypes or constructs are modest.

    Response:

    Statistical analyses have been added for immunoblot quantifications where appropriate, particularly in cases where differences between genotypes or constructs are modest.

    The naming of Mfi2 as a mitofissin is consistent with previous terminology introduced for Atg44, but the term remains relatively new. A brief clarification distinguishing "mitofissin" from the better-known "mitofusin" family in mammals would help avoid confusion for readers less familiar with yeast-specific nomenclature.

    Response:

    We have added a brief explanation of the term "mitofissin" to distinguish it from the mammalian "mitofusin" family in Introduction (Page 3, line 26-Page 4 line 1).

    Reviewer #3 (Significance (Required)):

    This is a strong and well-executed study that provides mechanistic insight into how mitochondrial fission is coordinated during mitophagy in yeast. A major strength is the identification and characterisation of Mfi2 as a previously unrecognised outer membrane fission factor acting in parallel with Dnm1 and in coordination with the intermembrane space protein Atg44. The genetic, imaging, and in vitro biochemical data are carefully integrated, and the authors are transparent about limitations, including open questions around the C-terminal domain of Mfi2, CL dependence, and the evolutionary conservation of mitofissins.

    The work makes a conceptual advance by showing that mitophagy-specific mitochondrial fission requires the cooperation of spatially separated factors acting from both the inside and outside of mitochondria, a mechanism that had not been fully appreciated. This study helps resolve previous contradictions regarding the dispensability of Dnm1 in mitophagy, thereby filling a gap in our understanding of organelle-specific fission. While the findings are focused on yeast, they raise broader questions about whether similar principles apply to higher eukaryotes (historically yeast research was always at the forefront of autophagy field).

    The study will be of interest to specialists in autophagy, mitochondrial dynamics, and yeast cell biology, as well as researchers working on membrane remodelling and organelle quality control. While the audience is primarily specialised, the conceptual insights will resonate more broadly in the cell biology community.

    I am an expert in mitophagy mechanisms in mammalian cells, and while not a specialist in yeast models, I found the study logical, rigorous, and of clear relevance to the broader autophagy field.

    Response:

    We are grateful for Reviewer #3’s recognition of the conceptual advance provided by our study and its relevance beyond yeast biology.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Responses to Reviewer #1:

    ・We performed in silico topology prediction of Atg44 and Mfi2 using TMHMM. This tool identified a weakly hydrophobic region of Mfi2 near the N-terminus but did not predict a definitive transmembrane domain (new Fig. EV1E) (Page 6, lines 8-9).

    Responses to Reviewer #3:

    ・We have added a brief explanatory sentence in the revised manuscript to clarify the methods and interpretation of the sodium carbonate extraction and proteinase K assays (Page 5, lines 23-25; Page 6, lines 1-3).

    ・Statistical analyses have been added for immunoblot quantifications where appropriate, particularly in cases where differences between genotypes or constructs are modest.

    ・We have added a brief explanation of the term "mitofissin" to distinguish it from the mammalian "mitofusin" family in Introduction (Page 3, line 26-Page 4, line 1).

    4. Description of analyses that authors prefer not to carry out

    Response to Reviewer #1 (Major 4):

    We will not include the WT strain as a control. See our response.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Furukawa et al. presents a well-structured and thorough study identifying Mfi2 as a novel mitochondrial outer membrane-resident fission factor required for mitophagy in Saccharomyces cerevisiae. The authors demonstrate that Mfi2, together with the inner membrane mitofissin Atg44 and the dynamin-related GTPase Dnm1, contributes to mitochondrial fragmentation during mitophagy. Importantly, they show that while Dnm1 is dispensable on its own, Mfi2 and Dnm1 act redundantly from the outer membrane to support Atg44-mediated fission. The data are robust, the figures are clear, and the mechanistic insight into how mitophagy-specific fission is achieved is of high relevance to the field of mitochondrial quality control. Overall, this is a logically constructed and convincing study with important implications for understanding compartment-specific mechanisms of mitochondrial fission during selective autophagy. The conclusions are largely well supported by the data. However, a few issues and points of clarification should be addressed before publication.

    Major Comments

    1. The observation that both Mfi2 and Atg44 require high cardiolipin (CL) content for membrane binding and fission in vitro is intriguing, especially given that CL is enriched in the inner membrane. The authors mention CL externalisation during mitophagy, but this connection could be made more explicit earlier in the manuscript. Furthermore, since the molecular mechanism of membrane interaction remains unresolved, I would strongly encourage the authors to undertake coarse-grained molecular dynamics simulations to explore how Mfi2 might interact with lipid bilayers of differing composition. This could clarify the role of CL and the potential structural contribution of the disordered C-terminal region.
    2. While the genetic and phenotypic data indicate that Mfi2 and Dnm1 act independently to support mitochondrial fission, the spatial and temporal organisation of their activity during mitophagy remains unclear. Do Mfi2 and Dnm1 colocalise at fission sites, or do they act at separate subdomains of the outer membrane? Live-cell imaging with fluorescently tagged Mfi2 and Dnm1, particularly during mitophagy induction, could help clarify whether these factors act in concert or at distinct locations and time points. This would also help determine whether their apparent redundancy reflects parallel mechanisms or functional compensation at shared sites. It would also be interesting to combine this with Atg44.

    Minor Comments

    1. The sodium carbonate extraction and proteinase K assays (Figure 1E-F) are standard but may not be familiar to all readers. A brief explanatory sentence clarifying what these methods reveal about membrane topology would improve accessibility.
    2. While immunoblot quantifications are shown throughout, it would be helpful to include statistical analysis where appropriate, especially in cases where differences between genotypes or constructs are modest.
    3. The naming of Mfi2 as a mitofissin is consistent with previous terminology introduced for Atg44, but the term remains relatively new. A brief clarification distinguishing "mitofissin" from the better-known "mitofusin" family in mammals would help avoid confusion for readers less familiar with yeast-specific nomenclature.

    Significance

    This is a strong and well-executed study that provides mechanistic insight into how mitochondrial fission is coordinated during mitophagy in yeast. A major strength is the identification and characterisation of Mfi2 as a previously unrecognised outer membrane fission factor acting in parallel with Dnm1 and in coordination with the intermembrane space protein Atg44. The genetic, imaging, and in vitro biochemical data are carefully integrated, and the authors are transparent about limitations, including open questions around the C-terminal domain of Mfi2, CL dependence, and the evolutionary conservation of mitofissins.

    The work makes a conceptual advance by showing that mitophagy-specific mitochondrial fission requires the cooperation of spatially separated factors acting from both the inside and outside of mitochondria, a mechanism that had not been fully appreciated. This study helps resolve previous contradictions regarding the dispensability of Dnm1 in mitophagy, thereby filling a gap in our understanding of organelle-specific fission. While the findings are focused on yeast, they raise broader questions about whether similar principles apply to higher eukaryotes (historically yeast research was always at the forefront of autophagy field).

    The study will be of interest to specialists in autophagy, mitochondrial dynamics, and yeast cell biology, as well as researchers working on membrane remodelling and organelle quality control. While the audience is primarily specialised, the conceptual insights will resonate more broadly in the cell biology community.

    I am an expert in mitophagy mechanisms in mammalian cells, and while not a specialist in yeast models, I found the study logical, rigorous, and of clear relevance to the broader autophagy field.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this study, the authors discover a mitochondrial fission factor, termed Mfi2, that promotes mitophagy efficiency and that functions in a partially redundant way with Dnm1 for the fission of mitochondrial outer membranes during mitophagy. The discovery helps to clarify why Dnm1 does not appear to be essential for fission mediated mitophagy by Dnm1. Mfi2 is structurally similar to the inner membrane fission factor Atg44 which is consistent with Mfi2's fission activity. The authors show that Mfi2 has membrane fission activity towards nanotubes in vitro, and that membrane binding is dependent of high levels of cardiolipin, a mitochondrially enriched lipid. In summary, the authors show that Mfi2 mediates mitochondrial outer membrane fission together with Drp1, whereas Atg44 mediates inner membrane fission, that together are necessary for mitophagy.

    Major:

    1. Figure 2: How do the expression levels of the Mfi2 constructs compare to the endogenous levels of the protein? This will help to gauge to what degree Mfi2 N66 overexpression is needed to achieve mitochondrial fragmentation in Atg44 delta cells and also the low level of mitophagy rescue that was observed.
    2. Figure 3A-B: The cardiolipin binding results in vitro are interesting but the concentration of cardiolipin is much lower on the outer membrane versus the inner membrane. Can the authors comment on whether the cardiolipin levels used on the nanotubes are relevant to that of the mitochondrial outer membrane where Mfi2 is located? Can the authors provide quantitative data for these experiments to help strengthen their conclusions? Can the authors also use purified MBP alone or a form of Mfi2 that cannot bind to membrane e.g. Mfi2-C33) as a control?
    3. Figure 4D: The protrusions are very difficult to visualize. Can the authors also provide zoomed in regions. Is the data representative from 3 or more independent experiments? Can the authors provide a graph of the quantitation to aid readers with analysis of the data?
    4. Figure 4D: It is fascinating to see the mitochondrial protrusion formation being dependent on autophagy factors but not mitochondrial fission factors. To help visualize this, can the authors image one of either Atg1, Atg8 to address whether phagophores are forming on the protrusions and if so where they are positionally located on the protrusion in control and/or mfi2,dnm1,atg44 triple mutant cells?

    Minor:

    1. Is it possible to target Atg44 to the mitochondrial outer membrane, either by attaching an OM anchor or using part of the N-terminus of Mfi2? This will help elucidate how Mfi2 reaches the outer membrane and whether Atg44 can be just as active on the outer membrane as long as it can access it.
    2. Are microtubules or actin required for the protrusions to form? Using the triple mutant cells that have a high proportion of protrusions, it could be tried to add cytoskeletal depolymerizing drugs such as nocodazole for microtubules or Latrunculin A or Latrunculin B for actin.

    Significance

    The discovery of Mfi2 as an outer membrane mitophagy fission factor is an exciting, and very important and significant contribution to the field. The data are in this study are clear and the conclusions are generally well supported by the experiments. This study appears to be suitable as a report style manuscript given that there is limited mechanistic analysis of Mfi2 activity. This does not affect the importance of the work, it just means that it is suited as a report of a significant discovery. Overall, this fills an important knowledge gap in solving the mystery behind which factors are involved in mitochondrial outer membrane fission during mitophagy, and provides a clarification why Dnm1 loss alone minimally affects mitophagy. This work will appeal to researchers interested in mitochondrial biology, the autophagy field, and cell biologists interested in organelle membrane dynamics, and is also broadly important and interesting to all cell biologists.

    Reviewer expertise: mitophagy mechanisms, cell biology of mitophagy, autophagy and autophagosome formation, mitochondrial biology including OXPHOS and mitochondrial dynamics

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Furukawa and colleagues identified Mfi2 as novel factor that promotes fragmentation and removal of damaged mitochondria by mitophagy. They report that parallel loss of Dnm1 and Mfi2 blocks mitophagy. Mfi2 acts on the outer membrane, while the previous found Atg44 functions in the intermembrane space. How the proteins cooperate remains unknown. This is an elegant study with high-quality data. The findings are interesting for a broad readership. There are some issues as outline below that should be solved.

    1. It remains unclear how Mfi2 is anchored into the outer mitochondrial membrane. Does it contain a transmembrane domain? The carbonate resistance indicates the presence of such transmembrane domain. However, the presented structures lack such membrane-spanning segment. This point should be clarified.
    2. How does Mfi2 cooperate with Dnm1? Is there any interaction between these proteins? Some further information could provide mechanistic insights into the function of Mfi2.
    3. The authors report a CL-dependent binding of Mfi2 to liposomes. Is the recruitment of Mfi2 to mitochondria impaired when CL-synthesis is blocked, e.g. in crd1delta mitochondria?
    4. Figure 4B: a wild-type control should be added.

    Significance

    The reported findings are interesting for a broad readership.

  5. Version published to 10.1101/2025.04.13.648642 on bioRxiv